Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “allosteric modulation”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 667 records · Page 37Linked to original sources
PubMed1988

Steroid modulation of the chloride ionophore in rat brain: structure-activity requirements, regional dependence and mechanism of action.

Further in vitro studies of steroids active at the gamma-aminobutyric acidA (GABAA) receptor regulated Cl- channel labeled by [35S]-t-butylbicyclophosphorothionate ([35S]TBPS) reveal additional structural requirements necessary for activity. Evaluation of selected steroids for activity against TBPS-induced convulsions show similar requirements for activity. Interestingly, steroids (e.g., 5 alpha-pregnan-3 alpha, 20 alpha-diol) were identified that have high potency but limited efficacy as modulators of [35S]TBPS binding. These characteristics are reminiscent of the clinically useful benzodiazepines (BZs) such as clonazepam. However, interactions between the prototypical anesthetic-barbiturate, sodium pentobarbital, and steroids active at the Cl- channel suggest that they do not share a common site of action as allosteric modulators of [35S]TBPS and BZ receptor binding. The most potent steroid evaluated, 5 alpha-pregnan-3 alpha-ol-20-one, modulates [35S]TBPS binding at low concentrations (IC50 approximately 17 nM) in a regionally dependent manner. All [35S]TBPS binding sites appear to be functionally coupled to a steroid "modulatory site." Because several of the active steroids are metabolites of progesterone, their ability to inhibit the binding of [3H]promegestrone to the cytosolic progestin receptor in rat uterus was evaluated. Those steroids showing potent activity at the GABAA receptor-Cl- ionophore were inactive at the intracellular progestin receptor. Such specificity coupled with their high potency provide additional support for the hypothesis that some of these steroids may be involved in the homeostatic regulation of brain excitability via the GABAA-BZ receptor complex.

Androstane-3,17-diol↗
PubMed1995

Influence of the estrus cycle on the discrimination of apparent neuroactive steroid site subtypes on the gamma-aminobutyric acidA receptor complex in the rat.

Estrus cycle-related changes in gamma-aminobutyric acid (GABA)A receptor complex (GRC) sensitivity to modulation by reduced progesterone metabolites is suggestive of a possible mechanism for maintaining brain homeostasis in the presence of fluctuating levels of these neuroactive metabolites. In addition, certain endogenously occurring pregnanediols are selective for apparent neuroactive steroid site subtypes discriminated by the progesterone metabolite 3 alpha-hydroxy-5 beta-pregnan-20-one (3 alpha,5 beta-P) on the GRC. Thus, it was of interest to evaluate the influence of gender and the estrus cycle on the ability of 3 alpha,5 beta-P and its 20-reduced analog 5 beta-pregnan-3 alpha,20 beta-diol to differentiate neuroactive steroid site subtypes. Neuroactive steroid modulation of [35S]t-butylbicyclophosphorothionate ([35S]TBPS) binding to the GRC in rats during estrus, diestrus and after ovariectomy (OVX) was measured in washed cortical P2 homogenates in the presence or absence of exogenous GABA. During diestrus, the inability of 5 beta-pregnan-3 alpha,20 beta-diol to allosterically modulate [35S]TBPS binding in the absence of GABA coincides with the inability of 3 alpha,5 beta-P to modulate [35S]TBPS binding with high potency. In contrast, the addition of GABA to the assay produced high potency inhibition of [35S]TBPS binding by each steroid. Remarkably, although findings in diestrus and OVX homogenates were no different from those observed in males, the proportions and IC50 values of the two sites discriminated by 3 alpha,5 beta-P in [35S]TBPS binding assays during the estrus phase were significantly different from male, OVX and diestrus rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Allosteric Regulation↗
PubMed2005

Group III metabotropic glutamate-receptor-mediated modulation of excitatory transmission in rodent substantia nigra pars compacta dopamine neurons.

Glutamate plays an important role in the regulation of dopamine neuron activity. In particular, the glutamatergic input from the subthalamic nucleus is thought to provide control over dopamine neuron firing patterns. The degeneration of dopamine neurons in the substantia nigra pars compacta (SNc) observed in Parkinson's disease (PD) is believed to be due to a complex interplay of factors, including oxidative stress and mitochondrial dysfunction. Although glutamate is not the primary cause of cell death in PD, there is evidence suggesting excessive glutamate release onto dopamine neurons may play a role in continued degeneration. Although many studies have focused on the role of glutamate in the SNc, little work has been directed at exploring the modulatory control of glutamate release in this region. Previous studies have found a high-potency inhibitory effect of nonselective group III mGluR agonist on glutamatergic transmission in the SNc. Using whole-cell patch-clamp methods and novel pharmacological tools, we have determined that mGluR4 mediates the group III mGluR modulation of excitatory transmission in the rat SNc. The group III mGluR-selective agonist l-(+)-2-amino-4-phosphonobutyric acid inhibits excitatory transmission in the SNc at low micromolar concentrations with a maximal inhibition occurring at 3 muM. This effect was potentiated by the mGluR4-selective allosteric modulator N-phenyl-7-(hydroxymino)cyclopropa[b]chromen-1a-carboxamide and was not mimicked by the mGluR8-selective agonist (S)-3,4-dicarboxyphenylglycine. Interestingly, in an attempt to employ knockout mice to confirm the role of mGluR4, we discovered an apparent species difference suggesting that in mice, both mGluR4 and mGluR8 modulate excitatory transmission in the SNc.

Aminobutyrates↗
PubMed1998

Effects of propofol and pentobarbital on ligand binding to GABAA receptors suggest a similar mechanism of action.

The GABAA receptor is allosterically modulated by a number of anesthetics and barbiturates. We have examined the effects of propofol and pentobarbital on the binding of the receptor agonist [3H]muscimol and the benzodiazepine modulators [3H]flunitrazepam and [3H]Ro15-4513 to bovine brain membranes. Both agents potentiated the binding of [3H]muscimol (5 nM), with EC50 values of 18.7 and 276 microM, respectively. The binding of [3H]muscimol is heterogeneous, suggesting the presence of both high (Kd approximately 10 nM) and low (Kd approximately 0.1-1.0 microM) affinity sites. The major effect of both propofol and pentobarbital was to increase the affinity of the lower affinity sites without changing the total binding capacity. In contrast, the steroid anesthetic alphaxalone did not affect the affinity of these sites, suggesting that this drug has distinct effects on the GABAA receptor. Propofol and pentobarbital also increased the binding of the benzodiazepine agonist [3H]flunitrazepam and decreased the binding of the inverse agonist [3H]Ro15-4513. The results of these studies demonstrate that propofol and pentobarbital modulate the binding of ligands to the GABAA receptor in a similar manner, suggesting that these drugs may have a common mechanism of action.

Allosteric Site↗
PubMed2004

Neurosteroidogenesis: relevance to neurosteroid actions in brain and modulation by psychotropic drugs.

Neurosteroids--i.e., steroid produced in brain ex novo or through metabolism of precursors--affect neuronal and brain functions through genomic and nongenomic mechanisms, depending on their molecular structure. Among neurosteroids, 3alpha-hydroxylated, 5alpha-reduced metabolites of progesterone (3alpha-hydroxy,5alpha-pregnan-20one/3alpha,5alpha-THP) and deoxycorticosterone (3alpha,21-dihydroxy,5alpha-pregnan-20one/3alpha,5alpha-THDOC) are positive allosteric modulators of gamma-aminobutyric acid (GABA) action at GABAA receptors. In rodents, a reduction of their endogenous brain concentrations rapidly lowers the potency of GABA in eliciting GABAA receptor-mediated inhibitory postsynaptic currents. This effect is related to anxiety-like behavior, increased aggression, and a reduced sensitivity to the loss of righting reflex induced by GABAA receptor agonist or positive modulators. Conversely, enhancement of 3alpha,5alpha-THP or 3alpha,5alpha-THDOC brain content results in anxiolysis, sedation/hypnosis, anticonvulsant, and anesthetic action. Different classes of psychotropic drugs--i.e., antidepressants, selected atypical antipsychotics, ethanol, gamma-hydroxybutyric acid--increase neurosteroid concentrations in brain, and these increases may be relevant to their pharmacological actions. Drug-induced increases of neurosteroids in rodent brain are often associated with elevation of their plasma content, such that alterations of plasma steroid concentrations are assumed to reflect parallel changes in brain. Nevertheless, brain neurosteroid concentrations are uneven across various regions, and the dose-dependence of their response to a pharmacological challenge shows brain-regional differences as well. These observations are consistent with the present knowledge on the distribution of steroidogenic enzymes in brain--they show not only a brain region, but also a cell-specific expression that may spatially and temporally determine the local concentrations of specific neurosteroids, either produced ex novo or through metabolism of steroid precursors that reach the brain through blood.

Anesthetics, Intravenous↗
PubMed1996

Neurosteroidal modulation of social isolation-induced decrease in pentobarbital sleep in mice.

Stressful manipulations are known to change the level of neurosteroids capable of interacting with GABAA receptor in the brain. To clarify the involvement of these neurosteroids in social isolation stress-induced decrease in pentobarbital sleep in mice, we examined the effects of 3 beta-hydroxy-5-pregnen-20-one-3-sulfate (pregnenolone sulfate, PS), a steroidal GABAA antagonist, and 3 alpha,21-dihydroxy-5 alpha-pregnan-20-one (allo-THDOC) and 3 alpha,21-dihydroxy-5 beta-pregnan-20-one (THDOC), positive allosteric modulators of the GABAA receptor, on the hypnotic activity of pentobarbital in socially isolated mice. Pentobarbital (50 mg/kg, i.p.)-induced sleep was significantly shorter in isolated mice than in group-housed mice and adrenalectomy had no effect on the decrease of pentobarbital sleep following social isolation. PS (5-10 mg/kg, i.p. or 12-24 nmol, i.c.v.) decreased pentobarbital sleep in group-housed mice in a dose-dependent manner without affecting the sleep in socially isolated mice. In contrast, allo-THDOC (14.9-37.4 nmol, i.c.v.) and THDOC (5-12.5 mg/kg, i.p. or 14.9-37.4 nmol, i.c.v.) reversed the pentobarbital sleep decreased by social isolation to the level in group-housed mice. These steroids had no effect on the pentobarbital sleep in group-housed mice. Such a reversing effect of THDOC in isolated mice was significantly blocked by PS (24 nmol, i.c.v.). Moreover, i.c.v. injection of yohimbine (30 nmol), methoxamine (200 nmol) and CRF (2.1 nmol) significantly decreased pentobarbital sleep in group-housed mice but not that in isolated mice. The effects of these drugs on pentobarbital sleep in group-housed mice were significantly attenuated by THDOC (12.5 mg/kg, i.p.). These results suggest that changes in the level of neurosteroids with ability to modulate GABAA receptor function are involved in social isolation-induced decrease in the hypnotic activity of pentobarbital in mice.

Adrenalectomy↗
PubMed1992

Purification and characterization of glucosamine-6-phosphate deaminase from dog kidney cortex.

Glucosamine-6-phosphate deaminase (EC 5.3.1.10) from dog kidney cortex was purified to homogeneity, as judged by several criteria of purity. The purification procedure was based on two biospecific affinity chromatography steps, one of them using N-epsilon-amino-n-hexanoyl-D-glucosamine-6-phosphate agarose, an immobilized analog of the allosteric ligand, and the other by binding the enzyme to phosphocellulose followed by substrate elution, which behaved as an active-site affinity chromatography. The enzyme is an hexameric protein of about 180 kDa composed of subunits of 30.4 kDa; its isoelectric point was 5.7. The sedimentation coefficient was 8.3S, and its frictional ratio was 1.28, indicating that dog deaminase is a globular protein. The enzyme displays positive homotropic cooperativity toward D-glucosamine-6-phosphate (Hill coefficient = 2.1, pH 8.8). Cooperativity was completely abolished by saturating concentrations of GlcNAc6P; this allosteric modulator activated the reaction with a typical K-effect. Under hyperbolic kinetics, a Km value of 0.25 +/- 0.02 mM for D-glucosamine-6-phosphate was obtained. Assuming six catalytic sites per molecule, kcat is 42 s-1. Substrate-velocity data were fitted to the Monod's allosteric model for the exclusive-binding case for both substrate and activator, with two interacting substrate sites. The Kdis for N-acetyl-D-glucosamine-6-phosphate was estimated at 14 microM.

Aldose-Ketose Isomerases↗
PubMed1984

Purification, molecular and kinetic properties of glucosamine-6-phosphate isomerase (deaminase) from Escherichia coli.

Glucosamine-6-phosphate isomerase (deaminase), (2-amino-2-deoxy-D-glucose-6-phosphate ketol isomerase (deaminating), EC 5.3.1.10) has been purified to homogeneity from Escherichia coli B as judged by several criteria of purity. The procedure included ammonium sulfate fractionation, anion-exchange chromatography and a biospecific affinity chromatography step with N-epsilon-amino-n- caproyl -D-glucosamine 6-phosphate bound to agarose as the ligand, the elution being performed with GlcNAc6 P. The enzyme appears to be an hexamer of about 178 kDa, composed of six subunits of 29 700 +/- 300 Da; the isoelectric point was 6.0-6.1 and the sedimentation constant 9.0 S. The amino-acid composition of the enzyme was determined and a value for E1%275 of 4.55 was calculated. The molecular activity was 1800 s-1 for the deamination reaction and 455 s-1 for the reaction of GlcN6 P formation. A positive homotropic cooperativity was found for both sugar substrates; it was stronger for GlcN6 P in the deamination reaction (Hill number 2.7 at pH 7.7). Ammonia behaved as a Michaelian substrate. Cooperativity was abolished by 0.1 mM GlcNAc6 P; this allosteric modulator activated the reaction in both directions, with a positive K-effect upon both sugar phosphates, but had no effect on Km for ammonia. The initial velocity patterns for the amination reaction were obtained under conditions of hyperbolic kinetics produced by GlcNAc6 P; the Km values for the allosteric substrates were determined under the same conditions, and their dependence upon pH was studied.

Aldose-Ketose Isomerases↗
PubMedDate not supplied

[Membrane receptors for estradiol--new way of biological action].

Classical action of steroid hormones, called genomic, includes binding to their intracellular receptor, require hours or days to occur and require transcriptional effects with subsequent modulation of protein expression. Some of the biological effects induced by steroids, and mainly by sex steroids, take place within seconds or few minutes, time far too fast to be due to the genomic changes. The rapid, nongenomic action of estradiol are attributed to membrane action, probably through variety of proteins present in cell membrane. The rapid effects of steroid hormones are manifold, ranging from activation of protein and tyrosine kinases, G proteins, and modulation of ion channels. The nongenomic way of action includes also non-direct control of processes of transcription and gene expression. There are at least three different way to interact with cell membrane. Steroids may change membrane fluidity, without binding to any known protein or receptor. Another way is allosteric modulation of non-specific for steroid hormones receptors, or structural and enzymatic protein present in cell membrane. Evidence suggests that the classical steroid receptors can be localized at the plasma membrane, triggering signals typical for G-proteins coupled receptors. Physiological significance of nongenomic action of steroids needs to be elucidated.

Animals↗
PubMed2002

[Effect of changes in extracellular pH on GABAA receptors in neurons].

Local pH values of both intra- and extracellular liquids can be regulated by a number of mechanisms including membrane transport and metabolism. It is known that the changes of extracellular pH accompanying physiological and pathological processes are sufficient to affect several important structures such as ionic channels, transporters, receptors etc. In particular, several reports indicate that GABAA receptor is strongly modulated by this factor. The effect of pH on these receptors strongly depend on the subtype of GABAA receptor (subunit composition). The application of ultrafast perfusion system allowed to explore the mechanisms of pH effect on GABAA receptors in neurons. It is concluded that changes in pH exert their effects by allosteric modulation of GABAA receptors.

Animals↗
PubMed2001

Mutagenesis of the GABA(A) receptor alpha1 subunit reveals a domain that affects sensitivity to GABA and benzodiazepine-site ligands.

We have mutated several amino acids in the region of the GABA(A) receptor alpha1 subunit predicted to form a small extracellular loop between transmembrane domains two and three to investigate its possible role in ligand sensitivity. The mutations were S275T, L276A, P277A, V279A, A280S and Y281F. Mutant alpha1 subunits were co-expressed with beta2 and gamma2 subunits in tsA201 cells or Xenopus oocytes. Binding studies revealed that the only mutation that significantly affected [3H]Ro15-4513 binding was the V279A substitution which reduced the affinity for this ligand. Electrophysiological examination of mutant receptors revealed that L276A, P277A and V279A displayed rightward shifts of their GABA concentration-response curves, the largest occurring with the L276A mutant. The impact of these mutations on allosteric modulation by benzodiazepine-site ligands was examined. V279A reduced the potency of both flunitrazepam and Ro15-4513 but, in each case, their efficacy was enhanced. A280S resulted in a decrease in flunitrazepam efficacy without affecting its potency. Additionally, P277A and A280S resulted in Ro15-4513 losing its inverse agonist effect at these receptors. These results suggest that a domain within this small extracellular loop between TMII-TMIII plays a role in determining the sensitivity of GABA(A) receptors to both GABA and benzodiazepine-site ligands.

Allosteric Regulation↗
PubMed2004

Designing human m1 muscarinic receptor-targeted hydrophobic eigenmode matched peptides as functional modulators.

A new proprietary de novo peptide design technique generated ten 15-residue peptides targeting and containing the leading nontransmembrane hydrophobic autocorrelation wavelengths, "modes", of the human m(1) muscarinic cholinergic receptor, m(1)AChR. These modes were also shared by the m(4)AChR subtype (but not the m(2), m(3), or m(5) subtypes) and the three-finger snake toxins that pseudoirreversibly bind m(1)AChR. The linear decomposition of the hydrophobically transformed m(1)AChR amino acid sequence yielded ordered eigenvectors of orthogonal hydrophobic variational patterns. The weighted sum of two eigenvectors formed the peptide design template. Amino acids were iteratively assigned to template positions randomly, within hydrophobic groups. One peptide demonstrated significant functional indirect agonist activity, and five produced significant positive allosteric modulation of atropine-reversible, direct-agonist-induced cellular activation in stably m(1)AChR-transfected Chinese hamster ovary cells, reflected in integrated extracellular acidification responses. The peptide positive allosteric ligands produced left-shifts and peptide concentration-response augmentation in integrated extracellular acidification response asymptotic sigmoidal functions and concentration-response behavior in Hill number indices of positive cooperativity. Peptide mode specificity was suggested by negative crossover experiments with human m(2)ACh and D(2) dopamine receptors. Morlet wavelet transformation of the leading eigenvector-derived, m(1)AChR eigenfunctions locates seven hydrophobic transmembrane segments and suggests possible extracellular loop locations for the peptide-receptor mode-matched, modulatory hydrophobic aggregation sites.

Algorithms↗
PubMed2002

The quaternary structure of the HisZ-HisG N-1-(5'-phosphoribosyl)-ATP transferase from Lactococcus lactis.

The N-1-(5'-phosphoribosyl)-ATP transferase (ATP-PRTase) encoded by the hisG locus catalyzes the condensation of ATP with PRPP, the first reaction in the biosynthesis of histidine. Unlike the homohexameric forms of the enzyme found in Escherichia coli and Salmonella typhimurium, the ATP-PRTase from Lactococcus lactis and a number of other bacterial species consists of two different polypeptides, both of which are required for catalytic activity (Sissler et al. (1999) Proc. Natl. Acad. Sci. 96, 8985-8990). The first of these is a truncated version of HisG that is approximately 100 amino acids shorter than the canonical versions. The second, HisZ, is a 328-residue version of a class II aminoacyl-tRNA synthetase catalytic domain that possesses no aminoacylation function. Here, the molecular mass and subunit composition of the L. lactis HisZ-HisG heteromeric ATP-PRTase is investigated using liquid chromatography, analytical ultracentrifugation, and quantitative protein sequencing. Individually, HisZ and HisG form inactive but stable dimers with association constants in the range of 2.5-3.3 x 10(5) M(-1). When both types of subunits are present, a quaternary octamer complex is formed with a sedimentation coefficient of 10.1 S. Incubation of this complex with ATP promotes a shift to 10.7 S. By contrast, incubation with the allosteric modulators AMP and histidine destabilizes the complex, resulting in a shift to multiple species in equilibrium with an average of 9.3 S. While this octameric structure is unique to both the phosphoribosyl transferases and the aminoacyl-tRNA synthetases, the change in sedimentation behavior elicited by substrates and inhibitors suggests the presence of allosteric regulatory mechanisms reminiscent of other multisubunit enzymes of metabolic importance.

Amino Acyl-tRNA Synthetases↗
PubMed2000

The neurosteroid allopregnanolone modulates oxytocin expression in the hypothalamic paraventricular nucleus.

Virgin, ovariectomized rats exposed to 2 wk of sequential estradiol (E(2)) and progesterone (P) followed by P withdrawal have increased hypothalamic oxytocin (OT) mRNA and peptide levels relative to sham-treated animals. This increase is prevented if P is sustained. In the central nervous system, P is metabolized to the neurosteroid allopregnanolone (3alpha-hydroxy-5alpha-pregnan-20-one), which exerts effects by acting as a positive allosteric modulator of GABA(A) receptor/Cl(-)-channel complexes. In the present study, ovariectomized rats that received sequential E(2) and P for 2 wk followed by P withdrawal were administered allopregnanolone at the time of P withdrawal. Hypothalamic and plasma allopregnanolone concentrations, serum E(2) and P concentrations, and hypothalamic OT mRNA levels were measured at death. Steroid-induced increases in OT mRNA were attenuated in animals treated with allopregnanolone at the time of P withdrawal. The results suggest that allopregnanolone plays an important modulatory role in steroid-mediated increases in hypothalamic OT.

Animals↗
PubMed2002

GABA(B) receptor-mediated increase of neurosteroids by gamma-hydroxybutyric acid.

Among the pharmacological actions of gamma-hydroxybutyric acid (GHB), some may involve GABA(A) receptor-mediated mechanisms. GHB, however, fails to directly interact with sites for agonists and modulators on the GABA(A) receptor complex. We hypothesized that, in vivo, GHB may interfere with GABA(A) receptor function by altering the brain concentrations of the neurosteroids 3 alpha-hydroxy-5 alpha-pregnan-20-one (allopregnanolone, AP) and 3 alpha,21-dihydroxy-5 alpha-pregnan-20-one (allotetrahydrodeoxycorticosterone, THDOC), positive allosteric modulators of GABA-gated chloride currents. In male Wistar rats, GHB dose-dependently (75-1000 mg/kg, i.p.) increased AP, THDOC and their precursors pregnenolone and progesterone in brain cortex and hippocampus. The increases of AP (4-5 fold) and THDOC (3-4 fold) elicited by 300 mg/kg GHB peaked between 30 and 90 min and abated by 180 min. The selective GABA(B) receptor antagonist SCH 50911 (50 mg/kg, i.p.) prevented the action of GHB, while the GABA(B) receptor agonist baclofen (5-10 mg/kg) mimicked it. NCS-382 (50 mg/kg, i.p.), the purported selective antagonist of the GHB receptor, failed to antagonize GHB, but at 300 mg/kg increased brain cortical neurosteroids to the same extent as 300 mg/kg GHB; coadministration of GHB and NCS-382, however, failed to yield an additive effect. These results strongly suggest that GHB, via a GABA(B) receptor-mediated mechanism, increases the brain concentrations of neurosteroids, whose properties as amplifiers of the GABA-gated chloride conductances may play a role in the GABA(A) receptor-mediated pharmacological actions of GHB.

Animals↗
PubMed1997

Sites of alcohol and volatile anaesthetic action on GABA(A) and glycine receptors.

Volatile anaesthetics have historically been considered to act in a nonspecific manner on the central nervous system. More recent studies, however, have revealed that the receptors for inhibitory neurotransmitters such as gamma-aminobutyric acid (GABA) and glycine are sensitive to clinically relevant concentrations of inhaled anaesthetics. The function of GABA(A) and glycine receptors is enhanced by a number of anaesthetics and alcohols, whereas activity of the related GABA rho1 receptor is reduced. We have used this difference in pharmacology to investigate the molecular basis for modulation of these receptors by anaesthetics and alcohols. By using chimaeric receptor constructs, we have identified a region of 45 amino-acid residues that is both necessary and sufficient for the enhancement of receptor function. Within this region, two specific amino-acid residues in transmembrane domains 2 and 3 are critical for allosteric modulation of both GABA(A) and glycine receptors by alcohols and two volatile anaesthetics. These observations support the idea that anaesthetics exert a specific effect on these ion-channel proteins, and allow for the future testing of specific hypotheses of the action of anaesthetics.

Alanine↗
PubMed2006

Discovery and characterization of orthosteric and allosteric muscarinic M2 acetylcholine receptor ligands by affinity selection-mass spectrometry.

Screening assays using target-based affinity selection coupled with high-sensitivity detection technologies to identify small-molecule hits from chemical libraries can provide a useful discovery approach that complements traditional assay systems. Affinity selection-mass spectrometry (AS-MS) is one such methodology that holds promise for providing selective and sensitive high-throughput screening platforms. Although AS-MS screening platforms have been used to discover small-molecule ligands of proteins from many target families, they have not yet been used routinely to screen integral membrane proteins. The authors present a proof-of-concept study using size exclusion chromatography coupled to AS-MS to perform a primary screen for small-molecule ligands of the purified muscarinic M2 acetylcholine receptor, a G-protein-coupled receptor. AS-MS is used to characterize the binding mechanisms of 2 newly discovered ligands. NGD-3350 is a novel M2-specific orthosteric antagonist of M2 function. NGD-3366 is an allosteric ligand with binding properties similar to the allosteric antagonist W-84, which decreases the dissociation rate of N-methyl-scopolamine from the M2 receptor. Binding properties of the ligands discerned from AS-MS assays agree with those from in vitro biochemical assays. The authors conclude that when used with appropriate small-molecule libraries, AS-MS may provide a useful high-throughput assay system for the discovery and characterization of all classes of integral membrane protein ligands, including allosteric modulators.

Acetylcholine↗
PubMed1988

Effects of thiol-reagents on [3H]alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid binding to rat telencephalic membranes.

The binding of [3H]alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid ([3H]AMPA), a ligand for the quisqualate subtype of excitatory amino acid receptors, was measured after chemical modifications of rat brain synaptic membranes. Treatment with oxidizing or thiol-alkylating agents did not modify [3H]AMPA binding, whereas treatment with several sulfhydryl reagents produced marked increases in binding. The involvement of free sulfhydryl groups in the regulation of the properties of [3H]AMPA binding sites was suggested by the specificity of p-chloromercuribenzoic acid (PCMB), its sulfonate analog p-chloromercuriphenyl-sulfonic acid (PCMBS), and HgCl2, plus the reversal of their effects after reduction with dithiothreitol. Pretreatment of synaptic membranes with the oxidizing agent 5,5'-dithiobis(2-nitrobenzoic acid) or the alkylating agent N-ethylmaleimide did not significantly affect [3H]AMPA binding but markedly reduced the enhancing effect of PCMBS. On the other hand, the increase in [3H]AMPA binding produced by PCMBS was not prevented by treatment with agonists such as quisqualate or L-glutamate and was produced equally well in resealed postsynaptic membranes with both lipophilic or nonlipophilic SH-reagents. Using filtration assays, two types of binding sites could be detected with high and low affinity for [3H]AMPA. Treatment with SH-reagents produced an increase in the Bmax for the high affinity component and a decrease in the Bmax for the low affinity component, accompanied by an increase in its affinity for the ligand. Using centrifugation assays, the same two types of sites could be detected under control conditions but treatment with SH-reagents produced an increase in affinity of the large component that prevented the analytical differentiation of the two sites. Treatment with SH-reagents also increased the binding of [3H] glutamate to the N-methyl-D-aspartate receptors but did not modify the binding of [3H]kainate to the kainate receptors or the strychnine-insensitive [3H]glycine binding. These results suggest that free sulfhydryl groups allosterically modulate the affinity of the quisqualate subtype of excitatory amino acid receptors and also indicate that different types of glutamate receptors might be differentially affected by chemical modification.

Allosteric Regulation↗